Delivering substance and drug delivery system using the same

ABSTRACT

An object of the present invention is to prepare substances which are excellent in delivery and enable drugs to be retained in a body effectively over a long period and to construct a drug delivery system using the substances. When the delivering substance which is obtained by reacting polyalkylene glycol or a reactive derivative thereof, a phospholipid and a drug with each other to form covalent bonds is administered systemically or topically, the substance is retained at a target site in a body for a long period, thereby making it possible to sustain drug efficacy over a long period by a single administration.

This application is a U.S. National Phase Application under 35 U.S.C.371 of International Application No. PCT/JP01/02882 filed Apr. 3, 2001.

TECHNICAL FIELD

The present invention relates to substances being excellent in deliverywhich are obtained reacting polyalkylene glycol or a reactive derivativethereof, a phospholipid and a drug with each other to form covalentbonds and to a drug delivery system which makes it possible to retainthe drug at a specific site of a body for a long period by administeringthe delivering substance systemically or topically.

BACKGROUND ART

Intraocular diseases such as diseases of a retina, an optic nerve or avitreous body are often intractable, and a development of an effectivetreatment method is eagerly desired. Though ocular diseases are mostgenerally treated by instillation of drugs, the drugs are hardlydelivered to the intraocular tissues such as a retina, rendering thetreatment of the intraocular diseases all the more difficult.

In view of this, a method of administering a drug directly to a specificsite of a body was attempted. For example, a technique for administeringa liposome or a microsphere containing a drug to intraocular tissuessuch as a vitreous body was reported (Published Japanese Translation ofPCT No. 508369/1994, Japanese Laid-open Patent Publication No.221322/1992 and the like).

However, it is not easy to control release of the drug by using theliposome. The liposome and the microsphere have large particlediameters. Accordingly, when they are administered to the intraoculartissues such as the vitreous body, transparency in the vitreous bodysometimes cannot be maintained.

On the other hand, when the drug is administered orally, the drug iseasily absorbed and metabolized in a stomach, a small intestine, a largeintestine, a liver. Accordingly, it is difficult to deliver the drug toa specific site to attain a concentration at which drug efficacy isexhibited.

From these facts, it is an important subject to prepare substances whichare excellent in delivery and enable drugs to be retained in a bodyeffectively over a long period and to create a drug delivery systemusing the substances.

DISCLOSURE OF THE INVENTION

Focusing attention on delivering substances and a drug delivery systemusing the delivering substances and studying them precisely, the presentinventors prepared substances being excellent in delivery which areobtained by reacting polyalkylene glycol or a reactive derivativethereof, a phospholipid and a drug with each other to form covalentbonds. Thus, it was found that the delivering substances and a systemicor topical drug delivery system using the delivering substances can beused for treatment of diseases at various sites of a body. When thedelivering substances are administered to the vitreous body, thedelivering substances are retained in a retina and a vitreous body for along period.

The present invention provides the delivering substances containingpolyalkylene glycol or a reactive derivative thereof, the phospholipidand the drug linked by covalent bonds, and the drug delivery system toadminister the substance systemically or topically. The presentinvention also provides a method of treatment comprising administeringsystemically or topically to a patient a pharmaceutically effectiveamount of the delivering substance containing polyalkylene glycol or thereactive derivative thereof, the phospholipid and the drug linked by thecovalent bonds solely or combined with a pharmaceutically acceptablecarrier or additive, and use of the substance.

When the delivering substance of the present invention is administeredsystemically or topically, the substance is retained at a target site ina body for a long period, thereby making it possible to sustain drugefficacy over a long period by a single administration.

The present invention relates to the delivering substances representedby the following general formula [1] and containing polyalkylene glycolor the reactive derivative thereof, the phospholipid and the drug linkedby the covalent bonds,(A

_(m)—X—Y—

B)_(n)  [1]wherein A and B, being the same or different, are residues of the drug,X is a residue of polyalkylene glycol or the reactive derivativethereof, Y is a phospholipid skeleton or a residue of the phospholipid,m is 0 or an integer of 1 or more, n is 0 or 1, at least one of m and nis not 0, and all of A, B, X and Y are linked by the covalent bonds,namely, “—” in the formula [1] stands for the covalent bond.

The polyalkylene glycol is a polymer containing a repeating unit[—O—alkylene—], and the alkylene can be substituted by lower alkyl orhydroxyl. Preferred examples of the polyalkylene glycol are polymersconsisting of C_(2—3) alkylene chains, and more preferred examplesthereof are polyethylene glycol and polypropylene glycol. The reactivederivative of the polyalkylene glycol is a derivative having at leastone chemically-modified terminal of the polyalkylene glycol so that thepolyalkylene glycol can be linked to the drug or the phospholipid by thecovalent bond. Preferred examples of the reactive derivative arederivatives having aminoalkyl, carboxyalkyl, mercaptoalkyl,hydrazidoalkyl, maleimidoalkyl, sulfonylalkyl, vinylsulfonylalkyl,vinylcarbonyl introduced into one or both of the terminals of thepolyalkylene glycol. More preferred examples of the reactive derivativeare derivatives having aminoethyl, aminopropyl, carboxymethyl,carboxyethyl, mercaptoethyl or hydrazidomethyl introduced into one orboth of the terminals.

When m is zero in the general formula [1], a OH group located at oneterminal of the polyalkylene glycol can be protected with alkyl, acyl orthe like.

The polyalkylene glycol or the reactive derivative thereof can be any ofstraight-chain, stellate and branched and can appropriately be selectedconsidering a concentration of the delivering substance at the targetsite, a period necessary for retaining the delivering substance at thetarget site, and the like. Plural drugs can be linked to one deliveringsubstance by covalent bonds by using the stellate or branchedpolyalkylene glycol or the reactive derivative thereof.

As a bonding form, the drug, the polyalkylene glycol (including thereactive derivative thereof) and the phospholipid are preferably linkedin the form of drug-polyalkylene glycol-phospholipid. They can also belinked in the form of polyalkylene glycol-phospholipid-drug ordrug-polyalkylene glycol-phospholipid-drug.

The plural drugs can be linked to the polyalkylene glycol by selecting asuitable polyalkylene glycol. Further, the plural drugs can be linked tothe delivering substance by linking the drug to the phospholipid.

A molecular weight of the polyalkylene glycol or the reactive derivativethereof constituting the delivering substance of the present inventionis not limited and can appropriately be selected considering a drugdelivery site of a body, the kind and properties of the drug forming thecovalent bond, a required concentration of the delivering substance, aperiod for retaining the delivering substance, and the like. Themolecular weight is usually 500 to 200,000, more preferably 1,000 to50,000.

Chemical structure of the drug linked to the polyalkylene glycol or thereactive derivative thereof by the covalent bond is not limited, and thedrug can have a functional group which can be linked to the polyalkyleneglycol or the reactive derivative thereof. Preferred examples of thedrug are ones having hydroxyl, carboxyl, carbonyl, amino, alkenyl. Thekind of the drugs is not limited so far as the drugs are systemic ortopical ones having therapeutic effects or preventive effects on variousdiseases. Examples of the drugs are anti-inflammatories,immunosuppressors, antivirals, antimicrobials, antimycotics, antitumors,nerve-protecting drugs, bloodflow-improving drugs, antiglaucomatousdrugs, analgesics, anesthetics, angiogenesis inhibitors, diagnosticagents. Examples of drugs to be used for treatment or prevention ofdiseases of a retina, an optic nerve, a vitreous body are drugs whichare effective for intraocular inflammation due to various causes, viralor bacterial infections, proliferative viteoretinopathy accompanied byproliferation of retinal cells, retinal neovasculaturigation, retinalhemorrhage due to various causes, retinal detachment or retinoblastoma.For example, anti-inflammatories such as betamethasone phosphate areused for treating inflammation accompanying an intraocular surgicaloperation. Immunosuppressors such as ciclosporin are used for treatingautoimmune uveitis. Antivirals such as ganciclovir are used for treatingviral infections. Antimicrobials such as ofloxacin are used for treatingpostoperative infections. Antitumors such as doxorubicin hydrochloride,carmustine, anti-VEGF and MMP inhibitors, ophthalmic diagnostic agentsare used for treating proliferative viteoretinopathy.

In order to link the polyalkylene glycol or the reactive derivativethereof to the drug by the covalent bond, they can be chemically reactedeach other, considering the functional group of the drug and thefunctional group of the polyalkylene glycol or the reactive derivativethereof. They can be linked by widely used methods. Though thepolyalkylene glycol itself can form the covalent bond, the reactivederivative thereof can much easily form the covalent bonds with variousdrugs. Since reactive derivatives of polyalkylene glycol having variousfunctional groups such as amino, thiol, carboxyl,succinimidylcarboxylate, epoxide, aldehyde, isocyanate, maleimide,acrylate and vinylsulfone are commercially available, the covalent bondscan be formed by chemically reacting these reactive derivatives with thedrug having the functional group.

Examples of the covalent bond formed in the delivering substances areester linkage, amide linkage, ether linkage, carbamate linkage, urealinkage, thiourea linkage, sulfide linkage, disulfide linkage, sulfonelinkage, carbonate linkage, a carbon—carbon bond. Delivering substanceshaving desired covalent bonds can be synthesized considering thefunctional group of the drug, the functional group of the polyalkyleneglycol or the reactive derivative thereof, a functional group of thephospholipid, a retention period at a diseased site in a body.

The phospholipid linked to the polyalkylene glycol or the reactivederivative thereof by the covalent bond is not limited and isexemplified by compounds represented by the following general formula[2] or salts thereof,

wherein R¹ and R², being the same or different, are hydrogen, alkyl,alkylcarbonyl, alkenyl or alkenylcarbonyl, and Z is aminoalkyl,diaminoalkyl, hydroxyalkyl or dihydroxyalkyl.

The phospholipid is not limited so far as the phospholipid has lowtoxicity and is excellent in safety. Examples of the phospholipid aresoybean lecithin, egg yolk lecithin, phosphatidylethanolamine,phosphatidylglycerol, phosphatidic acid, synthetic lecithin. Examples ofR¹ and R² in the compounds represented by the general formula [2] arealkylcarbonyl (alkanoyl) such as lauroyl, myristoyl, palmitoyl,stearoyl, oleoyl or linoleoyl, residues of the drugs. Examples of Z areaminoethyl, hydroxyethyl, 2,3-dihydroxypropyl.

The phospholipid preferably has a labile functional group in order tolink the polyalkylene glycol or the reactive derivative thereof to thephospholipid by the covalent bond. The functional group of thephospholipid is not limited and is exemplified by functional groupshaving lability such as amino in phosphatidylethanolamine, hydroxyl inphosphatidylglycerol and carboxyl in phosphatidylserine. A particularlypreferred phospholipid is phosphatidylethanolamine.

Examples of processes for linking the polyalkylene glycol or thereactive derivative thereof to the phospholipid by the covalent bond area process using an acid anhydride, a process using cyanuric chloride, aprocess using carbodliimide, a process using glutaraldehyde. The bestprocess can appropriately be selected among these processes to link acompound having the polyalkylene glycol or the reactive derivativethereof to the phospholipid by the covalent bond.

Chemical structure of the drug which can be linked to the phospholipidby the covalent bond is not limited, and the drug can have a functionalgroup which can be linked to the phospholipid. Examples of the drug arethe above-mentioned ones as the drugs linked to the polyalkylene glycolor the reactive derivative thereof by the covalent bond. The drug linkedto the phospholipid by the covalent bond can be the same as or differentfrom the drug linked to the polyalkylene glycol or the reactivederivative thereof by the covalent bond, and the drugs can appropriatelybe combined considering diseases, symptoms, drug efficacy and the like.

The delivering substances of the present invention can be prepared byvarious processes. For example, as shown by the following scheme, thecompound [A] is reacted with N-hydroxysuccinimide in the presence of acondensing agent (for example, N,N′-dicyclohexylcarbodiimide) to givethe active ester compound [B]. Next, the active ester moiety of thecompound [B] is reacted with a phospholipid having amino to give theamide compound [C]. t-Butoxycarbonyl introduced as a protecting group ofthe amide compound [C] is removed under an acidic condition to convertthe compound [C] into the amine compound [D]. This amine compound isreacted with an active carbonyl compound (for example, isothiocyanate)to give the delivering substance [E] of the present invention.

(wherein R¹ and R² have the same definitions as mentioned above, and tis an integer of 1 or more)

When the delivering substance of the present invention is administeredsystemically or topically, the delivering substance is retained at aspecific site of a body and is hardly metabolized. Accordingly, the drugis released gradually at the site, thereby exhibiting therapeutic andpreventive effects on diseases over a long period. The deliveringsubstance per se retained at the specific site of the body can alsoexhibit therapeutic and preventive effects on the diseases. Accordingly,the drug delivery system of the present invention particularly makes itpossible to treat the specific site of the body which has been difficultto treat so far, over a long period by a single administration.

The drug delivery system of the present invention can be used fortreating or preventing various diseases at the specific site of the bodyby administering the delivering substance of the present inventionsystemically or topically. Specific examples of the disease areinflammation due to various causes, viral or bacterial infections,immunodeficiency, tumor, proliferative viteoretinopathy accompanied byproliferation of retinal cells, retinal neovasculaturigation, opticneuropathy, retinal hemorrhage, retinal detachment, retinoblastoma.Various diagnoses can be conducted by administering systemically ortopically the delivering substance containing various diagnostic agentslinked by covalent bonds.

It is preferable to adjust a drug content in the delivering substance toa content so as to maintain an actual concentration of the drug with thelapse of time.

Advantageous effects of the present invention are described in detaillater in the section of the intraocular kinetic tests. Studying adelivering substance containing fluorescein linked by a covalent bond asa model drug for retentivity of the delivering substance in intraoculartissues (a vitreous body and a retina) after injection into the vitreousbody, the delivering substance of the present invention was proved to beretained in not only the vitreous body but also the retina over a longperiod (56 days or more). Injecting Dizocilpine (drug) per se, which isreported to have an optic nerve-protecting action, and a deliveringsubstance containing Dizocilpine linked by a covalent bond into vitreousbodies respectively and then comparing intraocular kinetics of the drug,it was elucidated that concentrations of the delivering substance of thepresent invention in the vitreous body, a retinochoroid and an opticnerve are 100 or more times higher than that in a case where Dizocilpineper se is used, and a disappearance half-life is also prolongedremarkably.

These test results show that various systemic or topical diseases can betreated effectively by selecting appropriately the drug linked by thecovalent bonds to the polyalkylene glycol or the reactive derivativethereof and/or the phospholipid of the present invention with a fewertimes of administration. When the drug delivery system of the presentinvention is used, the delivering substance can be retained efficientlyat the specific site of the body such as the retina, the optic nerve orthe vitreous body. Accordingly, it is possible to reduce an amount ofthe drug linked by the covalent bonds to the polyalkylene glycol or thereactive derivative thereof, the phospholipid, and an effect of reducingside-effects can also be exhibited.

Since the delivering substances of the present invention are retainedefficiently at the specific site in a body, the substances areparticularly effective for treating topical diseases. Their preparationforms are not limited and are exemplified by injections, infusions,tablets, ointments, emulsions, suspensions and the like. For example,various dosage forms and methods of administration such as eyedrops,injections, irrigations, iontophoresis and needleless injections can beused for ophthalmopathy. The delivering substances in the drug deliverysystem of the present invention can be formulated into preparation formssuited for methods of administration thereof (intraocular administrationand the like) by widely used processes. For example, in the case of theinjections, practical preparation examples thereof are described inExamples. The injections can be prepared by dissolving the deliveringsubstance prepared by the above-mentioned process in BSS (Balanced SaltSolution), a glycerin solution, a hyaluronic acid solution. Astabilizer, an isotonic agent, a buffer, a pH adjustor, a preservativecan optionally be added to the injections.

Examples of the stabilizer are edetic acid, disodium edetate. Examplesof the isotonic agent are glycerin, propylene glycol, polyethyleneglycol, sodium chloride, potassium chloride, sorbitol, mannitol .Examples of the buffer are citric acid, boric acid, sodiumhydrogenphosphate, glacial acetic acid, trometamol, ε-aminocaproic acid.Examples of the pH adjustor are hydrochloric acid, citric acid,phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide,sodium carbonate, sodium hydrogencarbonate. Examples of the preservativeare sorbic acid, potassium sorbate, benzalkonium chloride, benzethoniumchloride, p-hydroxybenzoate esters, sodium benzoate,dibutylhydroxytoluene, chlorobutanol, chlorhexidine gluconate.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing changes of concentration with time (21 days)in vitreous body tissues.

FIG. 2 is a graph showing changes of concentration with time (21 days)in retinochoroid tissues.

FIG. 3 is a graph showing changes of concentration with time (21 days)in optic nerve tissues.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples of the present invention are shown below, and they are intendedfor better understanding the present invention but are not to limit thescope of the present invention.

a. Preparation of Delivering Substances

Preparation Examples of delivering substances which can be used for adrug delivery system of the present invention are shown below.

EXAMPLE 1

(1) Delivering substance A, wherein

-   -   {circle around (1)} a compound wherein hydrogen of one terminal        OH group of polyethylene glycol (molecular weight: 5,000) is        substituted by thioureidoethyl and hydrogen of the other        terminal OH group is substituted by carbonylethyl,    -   {circle around (2)} fluorescein and    -   {circle around (3)} L-α-distearoylphosphatidylethanolamine are        linked by covalent bonds [chemical formula 6]

Methylene chloride (10 ml), chloroform (5 ml) and triethylamine (25 μl,0.18 mmol) were added to a mixture of an active ester wherein hydrogenof one terminal OH group of polyethylene glycol (molecular weight:5,000) was substituted by fluoresceinylthioureidoethyl and hydrogen ofthe other terminal OH group was substituted bysuccinimidyloxycarbonylethyl (Fluor-NHS-5k) [produced by Nippon Oils &Fats Co., Ltd.] (0.20 g, ca. 40 μmol) andL-α-distearoylphosphatidylethanolamine (61 mg, 82 μmol), and the wholewas stirred at room temperature overnight. Then, p-toluenesulfonic acid(40 mg, 0.21 mmol) was added to the reaction mixture, and the whole wasconcentrated under reduced pressure. 2-Propanol was added to theconcentrate, and the whole was stirred at room temperature for 30minutes. Then, precipitated crystals were filtered off, methanol (10 ml)was added to the crystals, and an insoluble matter was filtered out. Thefiltrate was concentrated under reduced pressure, 2-propanol was addedto the residue, and a precipitate was filtered off to give 151 mg of thedelivering substance A as orange crystals.

mp: 56.5-64.5° C.

IR (KBr,cm ⁻¹): 2886, 1741, 1611, 1468, 1344

(2) Delivering substance B, wherein

-   -   {circle around (1)} a compound wherein hydrogen of one terminal        OH group of polyethylene glycol (molecular weight: 5,000) is        substituted by thioureidoethyl and hydrogen of the other        terminal OH group is substituted by carbonylethyl,    -   {circle around (2)} fluorescein and    -   {circle around (3)} L-α-dioleoylphosphatidylethanolamine are        linked by covalent bonds    -   mp: 49.0-51.0° C.    -   IR (KBr,cm⁻¹): 2889, 1741, 1613, 1468, 1344

(3) Delivering substance C, wherein

-   -   {circle around (1)} a compound wherein hydrogen of one terminal        OH group of polyethylene glycol (molecular weight: 1,000) is        substituted by thioureidoethyl and hydrogen of the other        terminal OH group is substituted by carbonylethyl,    -   {circle around (2)} fluorescein and    -   {circle around (3)} L-α-distearoylphosphatidylethanolamine are        linked by covalent bonds    -   mp: 55.0-65.0° C.    -   IR (KBr,cm ⁻¹): 3313, 2917, 2850, 1748, 1617, 1540, 1468, 1349

(4) Delivering substance D, wherein

-   -   {circle around (1)} a compound wherein hydrogen of one terminal        OH group of polyethylene glycol (molecular weight: 10,000) is        substituted by thioureidoethyl and hydrogen of the other        terminal OH group is substituted by carbonylethyl,    -   {circle around (2)} fluorescein and    -   {circle around (3)} L-α-distearoylphosphatidylethanolamine are        linked by covalent bonds    -   mp: 55.0-60.0° C.    -   IR (KBr,cm⁻¹): 2885, 1745, 1614, 1468, 1343

(5) Delivering substance E, wherein

-   -   {circle around (1)} a compound wherein hydrogen of one terminal        OH group of polyethylene glycol (molecular weight: 5,000) is        substituted by thioureidoethyl and hydrogen of the other        terminal OH group is substituted by carbonylethyl,    -   {circle around (2)} fluorescein and    -   {circle around (3)} L-α-dimyristoylphosphatidylethanolamine are        linked by covalent bonds    -   mp: 65.0-75.0° C.    -   IR (KBr,cm ⁻¹): 2886, 1774, 1618, 1467, 1344

EXAMPLE 2

Delivering Substance F, wherein

-   -   {circle around (1)} a compound wherein hydrogen of both terminal        OH groups of polyethylene glycol (molecular weight: 5,000) is        substituted by carbonylethyl,    -   {circle around (2)} (±)        -3,4-dihydro-2-[5-methoxy-2-[3-[2-(3,4-methylenedioxy)phenoxyethyl-amino]propoxy]phenyl]-4-methyl-3-oxo-2H-1,4-benzothiazine        and    -   {circle around (3)} L-α-distearoylphosphatidylethanolamine are        linked by covalent bonds [chemical formula 7]

Chloroform (5 ml) was added to(±)-3,4-dihydro-2-[5-methoxy-2-[3-[2-(3,4-methylenedioxy)phenoxyethylamino]propoxy]phenyl]-4-methyl-3-oxo-2H-1,4-benzothiazinemonooxalate [a process for preparing this compound is disclosed JapaneseLaid-open Patent Publication No. 123181/1987.] (54 mg, 88 μmol), and theobtained mixture was stirred at room temperature. To the mixture wereadded triethylamine (0.04 ml, 0.3 mmol) and then an active ester whereinhydrogen of one terminal OH group of polyethylene glycol (molecularweight: 5,000) was substituted byL-α-distearoylphosphatidyloxyethylaminocarbonylethyl and hydrogen of theother terminal OH group was substituted by succinimidyloxycarbonylethyl(DSPE-NHS-5000) [produced by Nippon Oils & Fats Co., Ltd.] (0.30 g, ca.50 μmol). After one hour, p-toluenesulfonic acid monohydrate (0.20 g,1.1 mmol) was added to the reaction mixture, and the whole wasconcentrated under reduced pressure. 2-Propanol (20 ml) was added to theresidue, the whole was stirred at room temperature for 15 minutes, andthen an insoluble matter was filtered off to give 0.28 g of thedelivering substance F as colorless crystals.

-   -   mp: 51.7-56.1° C.    -   IR (KBr,cm³¹ ¹): 2887, 1742, 1467, 1113

EXAMPLE 3

Delivering Substance G, wherein

-   -   {circle around (1)} a compound wherein hydrogen of both terminal        OH groups of polyethylene glycol (molecular weight: 5,000) is        substituted by carbonylethyl,    -   {circle around (2)} [5R,        10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine        and    -   {circle around (3)} L-α-distearoylphosphatidylethanolamine are        linked by covalent bonds [chemical formula 8]

Methylene chloride (6.4 ml) was added to[5R,10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine[Dizocilpine] maleate (0.12 g, 0.36 mmol) under a nitrogen atmosphere,and the obtained mixture was stirred at room temperature. To the mixturewere added triethylamine (0.18 ml, 1.3 mmol) and then an active esterwherein hydrogen of one terminal OH group of polyethylene glycol(molecular weight: 5,000) was substituted byL-α-distearoylphosphatidyl-oxyethylaminocarbonylethyl and hydrogen ofthe other terminal OH group was substituted bysuccinimidyloxycarbonylethyl (DSPE-NHS-5000) [produced by Nippon Oils &Fats Co., Ltd.] (1.9 g, ca. 0.32 mmol), and the whole was stirredovernight. The reaction mixture was concentrated under reduced pressure,0.1 N hydrochloric acid (100 ml) was added to the concentrate, and thewhole was extracted with chloroform (100 ml) three times. The extractwas dried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by silica gel column chromatography,and the resulting crystals were filtered off to give 0.27 g of thedelivering substance G as colorless crystals.

mp: 52.4-56.9° C.

IR (KBr,cm⁻¹): 3434, 2885, 1742, 1715, 1467, 1344, 1149, 1120

b. Formulation Examples (injection)

A sterilized 2.6% glycerin solution (10 ml) was added to the deliveringsubstance G (30 mg), and the obtained mixture was warmed at 60° C. withstirring to give an injection in which the delivering substance G wasdissolved. Desired injections can be obtained by changing appropriatelythe kind of the delivering substance of the present invention and themixing ratio of the additive.

c. Intraocular Kinetic Tests by Fluorophotometry

Intraocular kinetic tests were conducted by the following methods usingthe delivering substances A and B containing a fluorescence.

Preparation of Delivering Substances:

Sterilized 2.6% glycerin solution (10 ml) was added to each of thedelivering substances A and B (36 mg), and the obtained mixture waswarmed to 60° C. with stirring to prepare an injection in which thedelivering substance A was dissolved and an injection in which thedelivering substance B was dissolved. The same procedure as mentionedabove was repeated except that fluorescein sodium was used instead ofthe delivering substances A and B to prepare a 10 μg/ml injectioncontaining fluorescein sodium for comparison.

Method of Administration and Method of Measurement:

1) A mixed solution containing an aqueous ketamine hydrochloridesolution (50 mg/ml) and an aqueous xylazine hydrochloride (50 mg/ml) ina ratio of 7:3 was administered intramuscularly to white rabbits toanesthetize.

2) A tropicamide (0.5%)/phenylephrine hydrochloride (0.5%) ophthalmicsolution was instilled into both eyes to cause mydriasis in the botheyes.

3) The both eyes were anesthetized with an oxybuprocaine hydrochloride(0.5%) ophthalmic solution.

4) Each of the above-mentioned injection was administered to the centerof a vitreous body from an ocular pars plana with a syringe equippedwith a 30G needle.

5) Intraocular fluorescence intensity was measured with afluorophotometry apparatus after the administration to the vitreous bodyat 1, 4, 7, 15, 35 and 56 days. A calibration curves were prepared,changes in concentration in the vitreous body and a retina weredetermined, and respective half-lives were calculated. Theabove-mentioned operations 1) and 2) were carried out before theintraocular fluorescence intensity was measured, too.

Results:

Table 1 shows half-lives of the delivering substances A and B andfluorescein sodium in the vitreous body, and Table 2 shows theirhalf-lives in the retina. The numerical values in Tables 1 and 2 are theaverage of three samples respectively.

TABLE 1 Test substance Half-life (days) Delivering substance A 7.0Delivering substance B 5.0 Fluorescein sodium <0.2 (The values in thetable were calculated by a moment method from data measured 1 to 35 daysafter the injection into the vitreous body.)

TABLE 2 Test substance Half-life (days) Delivering substance A 19.5Delivering substance B 16.5 Fluorescein sodium <0.1 (The values in thetable were calculated by the moment method from data measured 1 to 56days after the injection into the vitreous body.)Consideration:

Table 1 explicitly shows that the half-lives of the deliveringsubstances A and B in the vitreous body are 5.0 to 7.0 days, whereasthat of fluorescein sodium is only less than five hours. These resultsshow that the. delivering substances of the present invention prolongthe retention period in the vitreous body remarkably. Table 2 explicitlyshows that the half-lives of the delivering substances A and B in theretina are 16.5 to 19.5 days, whereas that of fluorescein sodium is onlyless than 2.4 hours. These results show that the delivering substancesadministered to the vitreous body migrate to the retina and are retainedthere for a long period.

d. Intraocular Kinetic Tests using Radioactive Isotopes

In order to study effects of the delivering substance G on retention inintraocular tissues (a vitreous body, a retina, an optic nerve and thelike), intraocular kinetic tests using radioactive isotopes wereconducted by the following methods.

Preparation of Drug Solutions:

The delivering substance G (9 mg) was weighed out and dissolved in a2.6% aqueous glycerin solution in a 5 ml measuring flask to adjust atotal amount to 5 ml. Into another test tube was introduced a 37 MBq/mlsolution of a compound prepared by labeling the delivering substance Gwith tritium [³H] (hereinafter referred to as “delivering substance G[³H]”) in toluene/ethanol (1:1) (200 μl), and toluene/ethanol wasevaporated under a nitrogen stream. The delivering substance G solution(5 ml) prepared previously was added to this test tube, and the obtainedmixture was stirred to prepare an administration solution.

On the other hand,[5R,10S]-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-iminemaleate (hereinafter referred to as “comparative substance X”) (0.96 mg)was weighed out and dissolved in a 2.6% aqueous glycerin solution in a10 ml measuring flask to adjust a total amount to 10 ml. Into anothertest tube was introduced a 37 MBq/ml solution of a compound prepared bylabeling the comparative substance X with tritium [³H] (hereinafterreferred to as “comparative substance X [³H]”) in ethanol (400 μl), andethanol was evaporated in a nitrogen stream. The comparative substance Xsolution (10 ml) prepared previously was added to this test tube, andthe obtained mixture was stirred to prepare an administration solution.Sterilized instruments were used in all the preparation.

Injection into Vitreous Body:

A mixed solution containing an aqueous ketamine hydrochloride solutionand an aqueous xylazine hydrochloride solution in a ratio of 7:3 wasinjected intramuscularly into Japanese white rabbits at a rate of 1ml/kg to anesthetize the rabbits. Next, each surface of both eyes wasanesthetized with an oxybuprocaine hydrochloride (0.5%) ophthalmicsolution, and then the administration solution of each test substance(100 μl/eye) was injected into a vitreous body with a 30G needle. Theinjection was carried out with the needle equipped with a stopper so asnot to introduce the needle into a retina. Table 3 shows concentrations,dosages of the respective administration solutions.

TABLE 3 Administered Adminstration Concentration Dosage radioactivitysolution (μmol/ml) (μl) (KBq/eye) Delivering 0.300 100 148 substance G[³H] Comparative 0.326 100 148 substance X [³H]Collection of Samples:

Prescribed days after the administration, an aqueous sodiumpentobarbital solution (50 mg/ml) (5 ml) was administered to ear veinsof Japanese white rabbits to sacrifice. Eyeballs were washed withphysiological saline (ca. 10 ml), then periphery of the eyeballs was cutfrom canthus or angulus oculi lateralis with a pair of scissors, and theeyeballs were enucleated. The eyeballs were washed with physiologicalsaline twice, and excessive water was wiped off with paper. Each bulbarconjunctiva was removed, and then an aqueous humor (ca. 0.2 ml) wascollected with a 1 ml syringe. Next, each eyeball was soaked in liquidnitrogen to freeze it, the eyeball was divided in two along its equatorportion with a razor, a vitreous body, a crystalline lens, the iris andciliary body, and a cornea were collected from an anterior portion, anda vitreous body, retinochoroid and an optic nerve were collected from aposterior portion.

Preparation of Samples for Measurement:

Wet weights of the collected vitreous body, retinochoroid and opticnerve were measured. After the measurement, they were dissolved with atissue-dissolving agent, and then to the obtained solution was added aliquid scintillator.

Preparation of Standard Radioactive Samples:

The delivering substance G [³H] administration solution and thecomparative substance X [³H] solution were diluted 1,000 timesrespectively to prepare standard radioactive samples.

Method of Determination:

Radioactivity concentrations of the prepared samples for measurement andstandard radioactive samples were measured with a liquid scintillationcounter. Radioactivity A per 1 ng of the test compound (dpm/pmol) wasdetermined from radioactivity of each standard radioactive sample, and aradioactivity concentration in each tissue was calculated by thefollowing equation.

Radioactivity concentration in tissue (pmol eq./g)=[{radioactivity ineach tissue sample (dpm)}/A (dpm/pmol)]{wet weight of tissue (g)}

Calculation of Pharmacokinetic Parameters:

An elimination half-life was calculated by the moment method from achange in concentration of each test substance in the intraocular tissuemeasured 1 to 21 days after the injection into the vitreous body.

Results:

FIGS. 1, 2 and 3 show respective changes in concentration of thedelivering substance G and the comparative substance X in the vitreousbody, the retinochoroid and the optic nerve after the injection into thevitreous body respectively. Tables 4 and 5 show half-lives of thedelivering substance G and the comparative substance X in the vitreousbody and a retina respectively. The numerical values in Tables 4 and 5are the average of three samples respectively.

TABLE 4 Test substance Half-life (days) Delivering substance G 3.3Comparative substance X 0.5

TABLE 5 Test substance Half-life (days) Delivering substance G 7.0Comparative substance X 0.6Consideration:

FIGS. 1 to 3 explicitly show that when the delivering substance G isadministered to the vitreous body, the delivering substance migrates toposterior segments such as the vitreous body, the retinochoroid and theoptic nerve and is retained at a high concentration over a long period.Tables 4 and 5 show that the half-life of the delivering substance isabout 6 to 10 times longer than that of the comparative substance X.

The delivering substances of the present invention are substances beingexcellent in delivery which are obtained by reacting polyalkylene glycolor a reactive derivative thereof, a phospholipid and a drug with eachother to form covalent bonds. The drug delivery system using thedelivering substances of the present invention can retain the deliveringsubstances in the posterior segments such as the vitreous body, theretina and the optic nerve for a long period. Accordingly, the drugdelivery system for administering the delivering substances systemicallyor topically makes it possible to treat or prevent various diseases atspecific sites of a body over a long period by a single administration.

INDUSTRIAL APPLICABILITY

The present invention provides substances being excellent in deliverywhich are obtained by reacting polyalkylene glycol or a reactivederivative thereof, a phospholipid and a drug with each other to formcovalent bonds, and a drug delivery system using the substances.

1. A method of treating a disease of a retina, an optic nerve or avitreous body comprising administering to an eye or eyes of a patient apharmaceutically effective amount of a delivering substance representedby the following formula [1] consisting essentially of a polyalkyleneglycol, a phospholipid and a drug linked by covalent bonds solely orcombined with a pharmaceutically acceptable carrier or additive,(A

_(m)—X—Y—

B)_(n)  [1] wherein A and B, being the same or different, are residuesof the drug; X is polyalkylene glycol or a reactive derivative thereofhaving aminoalkyl, carboxyalkyl, mercaptoalkyl, hydrazidoalkyl,maleimidoalkyl, sulfonyalkyl, vinylsulfonylalkyl or vinylcarbonylintroduced into one or both terminals of the polyalkylene glycol, Y is aresidue of the phospholipid, m is 0 or an integer of 1 or more, n is 0,1 or 2, at least one of m and n is not 0, and all of A, B, X and Y arelinked by the covalent bonds.
 2. A method of treating a disease of aretina, an optic nerve or a vitreous body comprising instilling in aneye or eyes of a patient a pharmaceuticallY effective amount of adelivering substance represented by the following formula [1] consistingessentially of a polyalkylene glycol, a phospholipid and a drug linkedby covalent bonds solely or combined with a pharmaceutically acceptablecarrier or additive,(A

_(m)—X—Y—

B)_(n)  [1] wherein A and B, being the same or different, are residuesof the drug, X is polyalkylene glycol or a reactive derivative thereofhaving aminoalkyl, carboxyalkyl, mercaptoalkyl, hydrazidoalkyl,maleimidoalkyl, sulfonylalkyl, vinylsulfonylalkyl or vinylcarbonylintroduced into one or both terminals of the polyalkylene glycol, Y is aresidue of the phospholipid, m is 0 or an integer of 1 or more, n is 0,1 or 2, at least one of m and ri is not 0, and all of A, B, X and Y arelinked by the covalent bonds.
 3. A method of treating a disease of avitreous body, a retina or an optic nerve comprising administering tothe vitreous body of a patient a pharmaceutically effective amount of adelivering substance represented by the following formula [1] consistingessentially of a polyalkylene glycol, a phospholipid and a drug linkedby covalent bonds solely or combined with a pharmaceutically acceptablecarrier or additive,(A

_(m)—X—Y—

B)_(n)  [1] wherein A and B, being the same or different, are residuesof the drug, X is polyalkylene glycol or a reactive derivative thereofhaving aminoalkyl, carboxyalkyl, mercaptoalkyl, hydrazidoalkyl,maleimidoalkyl, sulfonylalkyl, vinylsulfonylalkyl or vinylcarbonylintroduced into one or both terminals of the polyalkylene glycol, Y is aresidue of the phospholipid, m is 0 or an integer of 1 or more, n is 0,1 or 2, at least one of m and n is not 0, and all of A, B, X and Y arelinked by the covalent bonds.
 4. The method as claimed in claim 1,wherein the drug is an anti-inflammatory drug, an immunosuppressor, anantiviral drug, an antimicrobial drug, an antimycotic drug, an antitumordrug, a nerve-protecting drug, an ocular circulation improving drug, anantiglaucomatous drug, an analgesic, an anesthetic, an angiogenesisinhibitor or a diagnostic agent.
 5. The method as claimed in claim 2,wherein the drug is a drug for treatment of a disease of a retina, anoptic nerve or a vitreous body.
 6. The method as claimed in claim 2,wherein the drug is an anti-inflammatory drug, an immunosuppressor, anantiviral drug, an antimicrobial drug, an antimycotic drug, an antitumordrug, a nerve-protecting drug, an ocular circulation improving drug, anantiglaucomatous drug, an analgesic, an anesthetic, an angiogenesisinhibitor or a diagnostic agent.
 7. The method as claimed in claim 3,wherein the drug is an anti-inflammatory drug, an immunosuppressor, anantiviral drug, an antimicrobial drug, an antimycotic drug, an antitumordrug, a nerve-protecting drug, an ocular circulation improving drug, anantiglaucomatous drug, an analgesic, an aniogenesis inhibitor or adiagnostic agent.
 8. The method as claimed in claim 2, wherein the drugis a drug for treatment of a disease of a retina, an optic nerve or avitreous body.
 9. The method as claimed in claim 3, wherein the drug isa drug for treatment of a disease of a retina, an optic nerve or avitreous body.
 10. The method as claimed in claim 1, wherein the drug isselected from the group consisting of betamethasone phosphate,ciclosporin, ganciclovir, ofloxacin, doxorubicin hydrochloride andcarmustine.
 11. The method as claimed in claim 2, wherein the drug isselected from the group consisting of betamethasone phosphate,ciclosporin, ganciclovir, ofloxacin, doxorubicin hydrochloride andcarmustine.
 12. The method as claimed in claim 3, wherein the drug isselected from the group consisting of betamethasone phosphate,caclosporin, ganciclovir, ofloxacin, doxorubicin hydrochloride andcarmustine.